PROJECT SUMMARY Oral squamous cell carcinomas (OSCCs) have been proposed to exploit aspects of normal stem cell self- renewal and differentiation to drive cancer progression. Accordingly, activation of the Notch developmental pathway in OSCC has been implicated in cancer stem cell (CSC) function and drug resistance, suggesting that targeted inhibition of the pathway may be an effective therapeutic approach. However, the prevalence of Notch loss-of-function mutations in OSCC tumors also suggests that in certain contexts the Notch pathway may function as a tumor suppressor. The contexts in which Notch activation functions to promote or repress tumor progression are unknown and may dictate how Notch inhibitors can be used in the treatment of OSCC. We have identified two distinct but related OSCC populations with differential Notch pathway activation that may begin to address the distinct functions of the Notch pathway in relation to intra-tumor heterogeneity. Similar to other cancer types, we have isolated and characterized a subset of OSCC cells that reside in a quiescent, ?G0- like? state. These G0-like cells have low levels of total RNA and reactive oxygen species (ROS), are drug resistant, and activate the Notch pathway. Though G0-like cells lack upregulation of conventional CSC markers, including CD44 and ALDH1, they are able to transition to a slow-cycling CSC population identified by high expression of the histone demethylase JARID1B. Thus, Notch activation in G0-like cells may potentiate oral CSC function by promoting this transition to a JARID1Bhigh CSC state. As G0-like cells transition to the JARID1Bhigh state Notch pathway activation is reduced, suggesting that Notch pathway repression is required for JARID1Bhigh cells to exert stem cell-like function. Our preliminary data indicate that JARID1B functions to repress Notch signaling by regulating the expression of the Notch ligand JAG1. Therefore, our overall hypothesis is that Notch signals initiate entry of G0-like cells to a JARID1Bhigh state, where JARID1B- mediated suppression of the pathway is required for stem cell-like function. To test this hypothesis, we will first determine the functional role of Notch pathway activation in oral CSC maintenance (Aim 1). Here we will determine the effect of Notch activation or repression on the size and stem-like function of the G0-like population and characterize the requirement for Notch signaling in the G0-like to JARID1Bhigh transition. Additional mechanistic studies will be performed to elucidate the JARID1B-dependent regulation of Notch signaling and to determine the pathway?s role in the stem-cell like function of the JARID1Bhigh population (Aim 2). Taken together, these studies will define context-specific function for Notch signaling that will lead to much needed new therapeutic strategies to achieve lasting OSCC control. A carefully designed career-development and mentorship plan will facilitate the execution of the proposed studies.